The present invention relates to methods and compositions for delivering a polypeptide to a cell using poliovirus-based replicons. The invention relates to delivery of polypeptides that elicit an immune response in a subject. The invention relates to delivery of polypeptides that are capable of treating a disease condition in a subject. The invention further pertains to methods for generating cells that produce a non-poliovirus protein or fragment thereof.
Recent epidemiological data suggest that worldwide more than seventy percent of infections with human immunodeficiency virus (HIV) are acquired by heterosexual intercourse through mucosal surfaces of the genital tract and rectum. Most HIV vaccines developed to date have been designed to preferentially stimulate the systemic humoral immune system and have relied on immunization with purified, whole human immunodeficiency virus type 1 (HIV-1) and HIV-1 proteins (Haynes B F, 1993, Science 260:1279-1286), or infection with a recombinant virus or microbe which expresses HIV-1 proteins (McGhee J R et al., 1992, AIDS Res. Rev. 2:289-312). A general concern with these studies is that the method of presentation of the HIV-1 antigen to the immune system will not stimulate systemic and mucosal tissues to generate effective immunity at mucosal surfaces. Given the fact that the virus most often encounters a mucosal surface during sexual (vaginal or anal) transmission, a vaccine designed to stimulate both the systemic and mucosal immune systems is essential (McGhee J R et al., 1992, AIDS Res. Rev. 2:289-312; Forrest B D, 1992, AIDS Research and Human Retroviruses 8:1523-1525).
Worldwide, Helicobactor pylori is the most common cause of gastroduodenal ulcer and is an important risk factor for gastric cancer and gastric lymphoma (Novak M J et al., 1999, Vaccine 17(19):2384-2391). H. pylori infections can generally be treated with antibiotics.
However, drug-resistant variants exist and frequent use of antibiotics will exacerbate this problem by increasing the number of such variants. Thus, a vaccine for H. pylori would be of great benefit in developed and developing countries where H. pylori is endemic and gastric cancer is the second leading cause of cancer-related deaths. Eradication of H. pylori worldwide will likely require an effective therapeutic and prophylactic vaccine.
The use of neurotrophic viruses as vectors for targeted gene delivery to the central nervous system (CNS) has many applications for the development of new therapies for neurological diseases and spinal cord trauma.
Traumatic brain injury (TBI) affects nearly 200,000 people each year, most of them young men. Aggressive medical management has reduced the death rate, and currently, 75% of people survive a brain injury, but many are left with lasting cognitive and memory impairments that prevent their return to work or resumption of normal activities. Alterations in cognitive function remain a significant cause of long term morbidity after trauma to the central nervous system. Mild traumatic brain injury can result in cognitive deficits that are observed clinically and following experimental brain injury models (Dacey et al., 1993, in Cooper P R (ed): Head Injury. Baltimore. Williams and Wilkins pp. 159-182; Hicks, 1993, J. Neurotrauma 10: 405-414).
Most current therapies in clinical trials target prevention of neuronal injury and are aimed at early administration. This approach has not yet proven effective and must compete with intensive medical management of these very sick patients. Nerve growth factor belongs to the family of neurotrophic factors that regulate the survival and differentiation of nerve cells.
Thus, the unmet need for therapies for this population remains high.
One of the factors determining the degree to which elements of the central nervous system can recover from injury may be the availability of neurotrophic substances. Administration of various neuronal growth factors has been demonstrated to support neuronal cells in a variety of different models of central nervous system injury (Korsching S., 1993, J. Neurosci. 13:2739-2748; Maness et al., 1994, Neurosci. Biobehav. Rev. 18:143-159). Nerve growth factor remains the most extensively studied neurotrophic factor, and treatment with NGF has been shown to reduce cell death after neuronal injury (Kerr, JFR et al., 1991, in Tomei D L, Cope/FO (eds): Apoptosis The Molecular Basis of Cell Death, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press pp. 5-29; Frim D. M. et al., 1993, J. Neurosurg. 78: 267-273; Hagg T. et al., 1988, Exp. Neurol. 101: 303-312; Schumacher J. M. et al., 1991, Neuroscience 45: 561-570; Shigeno T. et al., 1991, J. Neurosci. 11: 2914-2919).
DeKosky S. T. et al., (1994, Exp. Neurol. 130:173-177), have demonstrated the presence of NGF in the cerebrospinal fluid of brain-injured human patients and NGF infusion can significantly improve the cognitive deficits normally associated with fluid-percussion brain trauma (Sinson G. et al., 1995, J. Neurochem. 65:2209-2216). Recent data indicates that NGF administration, in the acute, posttraumatic period following fluid-percussion brain injury, may have potential in improving post-traumatic cognitive deficits (Sinson et al., 1995, J. Neurochem. 65:2209-2216).
Nerve growth factor has been demonstrated to be a neurotrophic factor for forebrain cholinergic nerve cells that die during Alzheimer""s disease and with increasing age (PCT Publication WO 90/07341). Additionally, NGF can prevent the death of forebrain cholinergic nerve cells after traumatic injury and NGF has been reported to reverse the cognitive losses that occur with aging.
Intravenous application of certain nerve growth factors for the treatment of neuronal damage associated with ischemia, hypoxia or neurodegeneration has been described, however, the usefulness of such therapies is questionable given the presence of the blood brain barrier which prevents exposure of the damaged neuronal tissue to the intravenously administered NGF (PCT Publication Number WO 90/0882). Nerve growth factor can also be infused into the brain for treating neurodegenerative disorders, such as Parkinson""s disease, Alzheimer""s disease or Amyotrophic Lateral Sclerosis (ALS) by means of an implantable pump as described in PCT Publication Number WO 98/48723. In addition, NGF microencapsulation compositions having controlled release characteristics for use in promoting nerve cell growth, repair, survival, differentiation, maturation or function are described (PCT Publication Number WO 98/56426).
Poliovirus, a small RNA-virus of the family Picornaviridae, is an attractive candidate system for delivery of nucleic acids and proteins that may be useful in treating each of the foregoing maladies. Poliovirus-based replicons offer an attactive means to deliver antigens to the mucosal immune system and possibly treat or immunize against HIV or H. pylori infection. Additionally, poliovirus-based replicons offer an attractive means of delivering proteins, such as NGF, to neurons for alleviation or treatment of neurological disorders.
First, the live attenuated strains of poliovirus are safe for humans and are routinely administered to the general population in the form of the Sabin oral vaccine. Live or attenuated viruses have long been used to stimulate the immune system in a subject. A viral genome adapted for use in antigen delivery, therefore, should pose no greater health risk than that associated with administration of the attenuated vaccines alone.
Second, the pathogenesis of poliovirus is well-studied and the important features identified. The poliovirus is naturally transmitted by an oral-fecal route and is stable in the harsh conditions of the intestinal tract. Primary replication occurs in the oropharynx and gastrointestinal tract, with subsequent spread to the lymph nodes (Horstmann, D M et al., 1959, JAMA 170:1-8).
Upon entry into host cells, the RNA genome undergoes a rapid amplification cycle followed by an intense period of viral protein production. During this period, a poliovirus-encoded 2A protease arrests host cell cap-dependent protein synthesis by cleaving eukaryotic translation initiation factor 4GI (eIF4GI) and/or eIF4GII (Goldstaub D et al., 2000, Mol. Cell Biol. 20(4):1271-1277). Host cell protein synthesis may also be inhibited by proteolytic inactivation of transcription factors required for host cell gene expression (Das S et al., 1993, J. Virol. 67:3326-3331). The arrest of host cell protein synthesis allows poliovirus RNA, which does not require a 5xe2x80x2 cap for translation, to be selectively expressed over host transcripts. Moreover, arrested host cell protein synthesis is detrimental to the cell and may ultimately contribute to its death.
Third, the entire poliovirus genome has been cloned and sequenced and the viral proteins identified. An infectious poliovirus cDNA is also available which has allowed further genetic manipulation of the virus (Racaniello V R et al., 1981 Science 214(4542) 916-919). The genomic RNA molecule is 7433 nucleotides long, polyadenylated at the 3xe2x80x2 end and has a small covalently attached viral protein (VPg) at the 5xe2x80x2 terminus (Kitamura N et al., 1981, Nature 291:547-553; Racaniello V R et al., 1981, Proc. Natl. Acad. Sci. USA 78:4887-4891). Expression of the poliovirus genome occurs via the translation of a single protein (polyprotein) which is subsequently processed by virus encoded proteases (2A and 3C) to give the mature structural (capsid) and nonstructural proteins (Kitamura N et al., 1981, Nature 291:547-553; Koch F et al., 1985, The Molecular Biology of Poliovirus, Springer-Verlag, Vienna). Poliovirus replication is catalyzed by the virus-encoded RNA-dependent RNA polymerase (3DPol), which copies the genomic RNA to give a complementary RNA molecule, which then serves as a template for further RNA production (Koch F et al., 1985, The Molecular Biology of Poliovirus, Springer-Verlag, Vienna; Kuhn R J et al., 1987, in D J Rowlands et al. (ed.) Molecular Biology of Positive Strand RNA viruses, Academic Press Ltd., London). The translation and proteolytic processing of the poliovirus polyprotein is described in Nicklin M J H et al., 1986, Bio/Technology 4:33-42.
The viral RNA genome encodes the necessary proteins required for generation of new progeny RNA, as well as encapsidation of the new RNA genomes. In vitro, poliovirus is lytic, resulting in the complete destruction of permissive cells. Since the viral replication cycle does not include any DNA intermediates, there is no possibility of integration of viral DNA into the host chromosomal DNA.
The coding region and translation product of poliovirus RNA is divided into three primary regions (P1, P2, and P3). The mature poliovirus proteins are generated by a proteolytic cascade which occurs predominantly at Q-G amino acid pairs (Kitarnura N et al., 1981, Nature 291:547-553; Semler B L et al., 1981, Proc. Natl. Acad. Sci. USA 78:3763-3468; Semler B L et al., 1981, Virology 114:589-594; Palmenberg A C, 1990, Ann. Rev. Microbiol. 44:603-623). A poliovirus-specific protein, 3Cpro, is the protease responsible for the majority of the protease cleavages (Hanecak R et al., 1982, Proc.,Natl. Acad. Sci. USA 79:3973-3977; Hanecak R et al., 1984, Cell 37:1063-1073; Nicklin M J H et al., 1986, Bio/Technology 4:33-42; Harris K L et al., 1990, Seminars in Virol. 1:323-333). A second viral protease, 2APro, autocatalytically cleaves from the viral polyprotein to release P1, the capsid precursor (Toyoda H et al., 1986, Cell 45:761-770). A second, minor cleavage by 2APro occurs within the 3DPO1 to give 3Cxe2x80x2 and 3Dxe2x80x2 (Lee Y F et al., 1988, Virology 166:404-414). Another role of the 2Apro is the shut off of host cell protein synthesis by inducing the cleavage of a cellular protein required for cap-dependent translation (Bernstein H D et al., 1985, Mol. Cell Biol. 5:2913-2923; Krausslich H G et al., 1987, J. Virol. 61:2711-2718; Lloyd R E et al., 1988, J. Virol. 62:4216-4223).
Previous studies have established that the entire poliovirus genome is not required for RNA replication (Hagino-Yamagishi K et al., 1989, J. Virol. 63:5386-5392). Naturally occurring defective interfering particles (DIs) of poliovirus have the capacity for replication (Cole C N, 1975, Prog. Med. Virol. 20:180-207; Kuge S et al., 1986, J. Mol. Biol. 192:473-487). The common feature of the poliovirus DI genome is a partial deletion of the capsid (P1) region that still maintains the translational reading frame of the single polyprotein through which expression of the entire poliovirus genome occurs. In recent years, the availability of infectious cDNA clones of the poliovirus genome has facilitated further study to define the regions required for RNA replication (Racaniello V R et al., 1981 Science 214(4542) 916-919). Specifically, the deletion of 1,782 nucleotides of P1, corresponding to nucleotides 1174 to 2956, resulted in an RNA which can replicate upon transfection into tissue culture cells (Hagino-Yamagishi K et al., 1989, J. Virol. 63:5386-5392).
Fourth, previous studies using the attenuated vaccine strains of poliovirus have demonstrated that a long-lasting systemic and mucosal immunity is generated after administration of the vaccine (Sanders D Y et al., 1974, J. Ped. 84:406-408; Melnick J, 1978, Bull. World Health Organ. 56:21-38; Racaniello V R et al., 1981 Science 214(4542) 916-919; Ogra P L, 1984, Rev. Infect. Dis. 6:S361-S368).
In 1991, a group of researchers reported the construction and characterization of chimeric HIV-1-poliovirus genomes (Choi W S et al., 1991, J. Virol. 65(6):2875-2883). Segments of the HIV-1 proviral DNA containing the gag, pol, and env gene were inserted into the poliovirus cDNA so that the translational reading frame was conserved between the HIV-1 and poliovirus genes. The RNAs derived from the in vitro transcription of the genomes, when transfected into cells, replicated and expressed the appropriate HIV-1 protein as a fusion with the poliovirus P1 protein (Choi W S et al., 1991, J. Virol. 65(6):2875-2883). However, since the chimeric HIV-1-poliovirus genomes were constructed by replacing poliovirus capsid genes with the HIV-1 gag, pol, or env, genes, the chimeric HIV-1-genomes were not capable of encapsidation after introduction into host cells (Choi W S et al., 1991, J. Virol. 65(6):2875-2883). Furthermore, attempts to encapsidate the chimeric genome by cotransfection with the poliovirus infectious RNA yielded no evidence of encapsidation (Choi W S et al., 1991, J. Virol. 65(6):2875-2883).
In 1992, another group of researchers reported the encapsidation of a poliovirus replicon which incorporated the reporter gene, chloramphenicol acetyltransferase (CAT), in place of the region coding for capsid proteins VP4, VP2, and a portion of VP3 in the genome of poliovirus type 3 (Percy N et al., 1992, J. Virol. 66(8):5040-5046). Encapsidation of the poliovirus replicon was accomplished by first transfecting host cells with the poliovirus replicon and then infecting the host cells with type 3 poliovirus (Percy N et al., 1992, J. Virol. 66(8):5040-5046). The formation of the capsid around the poliovirus genome is believed to be the result of interactions between capsid proteins and the poliovirus genome. Therefore, it is likely that the yield of encapsidated viruses obtained by Percy et al. consisted of a mixture of encapsidated poliovirus replicons and encapsidated nucleic acid from the type 3 poliovirus. The encapsidated type 3 poliovirus most likely represents a greater proportion of the encapsidated viruses than does the encapsidated poliovirus replicons. The Percy et al. method of encapsidating a poliovirus replicon is, therefore, an inefficient system for producing encapsidated replicon.
Accordingly, it would be desirable to provide a method of encapsidating a recombinant poliovirus genome which results in a stock of encapsidated viruses substantially composed of the recombinant poliovirus genome. Such a method would provide for efficient production of encapsidated poliovirus nucleic acid for use in compositions for stimulating an immune response to non-poliovirus proteins encoded by the replicon genome as well as for compositions for delivering non-poliovirus proteins to neuronal tissue.
The present invention relates to poliovirus-based replicons. Replicons of the invention lack at least a portion of a sequence necessary for poliovirus encapsidation and cannot produce new encapsidated vectors following entry into a cell. However, replicons of the invention are fully capable of RNA replication (amplification) upon introduction into cells and optionally comprise non-poliovirus translatable sequences.
The present invention relates to methods for delivering a therapeutic polypeptide, or fragment thereof, to a cell by contacting the cell with a composition comprising poliovirus-based replicons. Replicons of the invention can comprise an expressible polynucleotide encoding a therapeutic polypeptide or fragment thereof. In some embodiments of the invention, the cell is a cell of the central nervous system, e.g. a neuronal cell. In some embodiments of the invention, the cell containing the replicon is transplanted into a recipient animal.
The invention also pertains to methods for delivering a therapeutic polypeptide, or fragment thereof, to a subject by administering to the subject a composition comprising a replicon encoding the therapeutic polypeptide or fragment thereof in an amount sufficient to obtain expression of the polypeptide. In particular, the therapeutic polypeptide or fragment thereof is a growth factor, cytokine (e.g., tumor necrosis factor alpha), receptor, transcriptional regulator, oncogene, tumor suppressor, or polypeptide with an enzymatic activity. The therapeutic polypeptide may also be a Helicobacter pylori polypeptide. In addition, the therapeutic polypeptide or fragment thereof is an immunogenic polypeptide which induces an immune response in the subject.
The invention pertains to methods of treating a subject with a disease, or likely to have a disease, comprising administering to the subject the replicon composition of the invention such that an amount of the therapeutic polypeptide or fragment thereof, effective to alleviate the symptoms of disease or prevent disease is expressed in the subject. The methods and compositions of the present invention are useful both in prophylaxis and in therapeutic treatment of disease, e.g. a neurodegenerative disease, or an infectious disease. The present invention also pertains to the use of encapsidated RNA replicons derived from type 1 poliovirus for the treatment of cellular proliferative and/or differentiative disorders, such as a cancer (e.g. carcinoma, sarcoma, lymphoma or leukemia).
The invention further pertains to methods for generating cells that produce a non-poliovirus protein or fragment thereof. In some embodiments of the invention, the method comprises (a) contacting cells with encapsidated replicons having an expressible non-poliovirus nucleic acid substituted for a nucleic acid which encodes at least a portion of a protein necessary for encapsidation and (b) maintaining the cells under conditions appropriate for introduction of the replicons into the host cells. The resultant cells are capable of producing a non-poliovirus protein or fragment thereof. In some embodiments of the invention, the method comprises (a) contacting cells with (i) encapsidated replicons having an expressible non-poliovirus nucleic acid substituted for a nucleic acid which encodes at least a portion of a protein necessary for encapsidation and (ii) a replicon encapsidation vector that encodes and directs expression of at least a portion of a protein necessary for replicon encapsidation, but which lacks an infectious poliovirus genome; and (b) maintaining the cells under conditions appropriate for introduction of the replicons and the encapsidation vector into the host cells. The resultant cells are capable of producing a non-poliovirus protein or fragment thereof.
In some embodiments of the invention, cells modified according to a method of the invention are introduced into a subject. The introduced cells produce the non-poliovirus replicon-encoded protein in said subject. In some embodiments of the invention, the cells used are first removed from a subject, subjected to one of the foregoing methods, and reintroduced into the same or another subject.